Proposal is designed to elucidate pharmacologic mechanisms whereby endotoxin alters vascular reactivity. Endotoxin exposure in vivo or in vitro impairs contractile responses in isolated aorta principally by inducing the expression of an (abnormal) nitric oxide (NO) synthase activity. It acts separately on endothelium to inhibit the elaboration of endothelium-derived relaxing factor (EDRF) in response to vasorelaxant agonists. Proposed studies, performed in cultured endothelial and vascular smooth muscle cells, isolated conduit and resistance size arteries, will determine cellular mechanisms mediating these vascular effects of endotoxin. Protocols will use measures of isometric contraction and relaxation, superfusion bioassay of EDRF production, fluorescence measurement of cytosolic calcium, responses of permeabilized arteries, and of microvascular tissue to explore the hypotheses that: 1) Endotoxin exposure modulates the activity of specific endothelial signal-transduction systems impairing EDRF synthesis by limiting the rise in endothelial cytosolic free calcium in response to vasorelaxant agonists, 2) Endotoxin impairs vascular smooth muscle contractile responses, principally via an NO-dependent mechanism, both by limiting the availability of cytosolic calcium as well as by impairing the calcium sensitivity of the contractile apparatus, 3) Endotoxin induces similar changes in microvascular endothelial and smooth muscle function, contributing to its systemic hemodynamic effect. Sepsis, or endotoxin administration, often results in pressor-refractory hypotension due primarily to low systemic vascular resistance and to organ failure which may be related to an impairment in tissue oxygen utilization by disordered regulation of blood flow distribution. Results of these studies will clarify mechanisms responsible for endotoxin-induced changes in both vasoconstriction and vasodilation, providing insights into pathophysiology of hemodynamic changes in sepsis which may be exploited therapeutically.